AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

Tiam-1, a GEF for Rac1, plays a critical role in metformin-mediated glucose uptake in C2C12 cells

Metformin is known to stimulate glucose uptake, but the mechanism for this action is not fully understood. In this study, AMPK activators (AICAR and metformin) increased the expression of T-lymphoma invasion and metastasis-inducing protein-1 (Tiam-1), a Rac1 specific guanine nucleotide exchange factor (GEF), mRNA and protein in skeletal muscle C2C12 cells. Metformin increases the serine-phosphorylation of Tiam-1 by AMPK and induces interaction between Tiam-1 and 14-3-3. Pharmacologic inhibition of AMPK blocks this interaction, indicating that 14-3-3 may be required for induction of Tiam-1 by AMPK. Metformin also increases the phosphorylation of p21-activated kinase 1 (PAK1), a direct downstream target of Rac1, dependent on AMPK. Tiam-1 is down-regulated at high glucose concentrations in cultured cells and in the db/db mouse model of hyperglycemia. Furthermore, Tiam-1 knock-down blocked metformin-induced increase in glucose uptake. These findings suggest that metformin promotes cellular glucose uptake in part through Tiam-1 induction.     

AMPKα2 translocates into the nucleus and interacts with hnRNP H: Implications in metformin-mediated glucose uptake

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cytoplasmic protein that plays a critical role in the maintenance of energy homeostasis. However, its role in the nucleus is still largely unknown. Here, we showed that AMPKα2 translocated into the nucleus during muscle differentiation. We also showed that upon treatment with 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR), an AMPK activator, AMPK rapidly translocated into the nucleus in rat myoblast L6 cells. On the other hand, the AMPKα2 phosphorylation-defective mutant did not translocate into the nucleus. Knockdown of AMPKα2 suppressed the differentiation-induced expression of myogenin, a differentiation marker. A physiological AMPK activator, metformin, also induced the translocation of AMPKα2 into the nucleus. Both inhibition and knockdown of AMPKα2 suppressed metformin-mediated glucose uptake. In addition, AMPKα2 was shown to directly interact with the heterogeneous nuclear ribonucleoprotein H (hnRNP H). AICAR treatment increased the phosphorylation of hnRNP H. Metformin increased the interaction between AMPKα2 and hnRNP H in the nucleus. Knockdown of hnRNP H blocked metformin-induced glucose uptake. In summary, these results demonstrate that AMPKα2 translocates into the nucleus via phosphorylation, AMPKα2 interacts with and phosphorylates hnRNP H in the nucleus, and such a protein–protein interaction modulates metformin-mediated glucose uptake.     

In vitro evaluation of effects of metformin on morphine and methadone tolerance through mammalian target of rapamycin signaling pathway

The chronic use of opioids leads to tolerance, psychological, and physical dependence that limits their use as an effective long-term pain control. Several studies have shown that mammalian target of rapamycin (mTOR) plays a crucial role in the development of opioid tolerance. Metformin activates 5′ adenosine monophosphate-activated protein kinase (AMPK) which directly suppresses the mTOR complex 1 signaling pathway. On the other hand, metformin can also inhibit mTOR directly and in an AMPK-independent manner. Thus, in the current study, we aimed to investigate the effects of metformin on the development of morphine and/or methadone-induced tolerance in human glioblastoma (T98G) cell line. We examined the effects of chronic treatment of morphine and/or methadone in the presence or absence of metformin with or without AMPK inhibitor (dorsomorphin hydrochloride) on levels of nitric oxide and cyclic adenosine monophosphate (cAMP), phosphorylated and dephosphorylated ribosomal protein S6 kinase β-1 (S6K1) and 4E-binding protein 1 (4E-BP1) in T98G cells. Pretreatment of cells with metformin (40 µM) with or without AMPK inhibitor (dorsomorphin hydrochloride; 1 µM) before adding of morphine (2.5 µM) or methadone (1 µM) revealed a protective effects on the development of opioid tolerance. Prior administration of metformin reversed the elevation of nitric oxide levels induced by morphine (p < 0.001) and methadone (p < 0.001) and also prevented the raise of cAMP levels induced by morphine in T98G cells (p < 0.05). Contribution of mTOR signaling pathway in metformin-induced effect was shown by the inhibition of phosphorylation of S6K1 and 4E-BP1, the downstream targets of mTOR. mTOR activation suppresses opioid-induced antinociception, and its activity has also been increased during opioid tolerance.     

UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

Background

AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that is activated when cells experience energy deficiency and conversely suppressed in surfeit of energy supply. AMPK activation improves insulin sensitivity via multiple mechanisms, among which AMPK suppresses mTOR/S6K-mediated negative feedback regulation of insulin signaling.

Results

In the present study we further investigated the mechanism of AMPK-regulated insulin signaling. Our results showed that 5-aminoimidazole-4-carboxamide-1 ribonucleoside (AICAR) greatly enhanced the ability of insulin to stimulate the insulin receptor substrate-1 (IRS1)-associated PI3K activity in differentiated 3T3-F442a adipocytes, leading to increased Akt phosphorylation at S473, whereas insulin-stimulated activation of mTOR was diminished. In 3T3-F442a preadipocytes, these effects were attenuated by expression of a dominant negative mutant of AMPK α1 subunit. The enhancing effect of ACIAR on Akt phosphorylation was also observed when the cells were treated with EGF, suggesting that it is regulated at a step beyond IR/IRS1. Indeed, when the cells were chronically treated with AICAR in the absence of insulin, Akt phosphorylation was progressively increased. This event was associated with an increase in levels of phosphatidylinositol -3,4,5-trisphosphate (PIP3) and blocked by Wortmannin. We then expressed the dominant negative mutant of PTEN (C124S) and found that the inhibition of endogenous PTEN per se did not affect phosphorylation of Akt at basal levels or upon treatment with AICAR or insulin. Thus, this result suggests that AMPK activation of Akt is not mediated by regulating phosphatase and tensin homologue (PTEN).

Conclusion

Our present study demonstrates that AMPK exerts dual effects on the PI3K pathway, stimulating PI3K/Akt and inhibiting mTOR/S6K.     

AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the alpha subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPKalpha or a kinase-inactive AMPKalpha, respectively. An insulin-resistant state was induced by exposing cells to 30 mm glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3alpha/beta. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPKalpha. The kinase-inactive AMPKalpha increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPKalpha negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.     

Resistin promotes cardiac hypertrophy via the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) and c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.     

Metformin protects against insulin resistance induced by high uric acid in cardiomyocytes via AMPK signalling pathways in vitro and in vivo

High uric acid (HUA) is associated with insulin resistance (IR) in cardiomyocytes. We investigated whether metformin protects against HUA-induced IR in cardiomyocytes. We exposed primary cardiomyocytes to HUA, and cellular glucose uptake was quantified by measuring the uptake of 2-NBDG, a fluorescent glucose analog. Western blot was used to examine the levels of signalling protein. Membrane of glucose transporter type 4 (GLUT4) was analysed by immunofluorescence. We monitored the impact of metformin on HUA-induced IR and in myocardial tissue of an acute hyperuricaemia mouse model established by potassium oxonate treatment. Treatment with metformin protected against HUA-reduced glucose uptake induced by insulin in cardiomyocytes. HUA directly inhibited the phosphorylation of Akt and the translocation of GLUT4 induced by insulin, which was blocked by metformin. Metformin promoted phosphorylation of AMP-activated protein kinase (AMPK) and restored the insulin-stimulated glucose uptake in HUA-induced IR cardiomyocytes. As a result of these effects, in a mouse model of acute hyperuricaemia, metformin improved insulin tolerance and glucose tolerance, accompanied by increased AMPK phosphorylation, Akt phosphorylation and translocation of GLUT4 in myocardial tissues. As expected, AICAR, another AMPK activator, had similar effects to metformin, demonstrating the important role of AMPK activation in protecting against IR induced by HUA in cardiomyocytes. Metformin protects against IR induced by HUA in cardiomyocytes and improves insulin tolerance and glucose tolerance in an acute hyperuricaemic mouse model, along with the activation of AMPK. Consequently, metformin may be an important potential new treatment strategy for hyperuricaemia-related cardiovascular disease.     

Activation of imidazoline I-2B receptor by metformin to increase glucose uptake in skeletal muscle

Metformin (dimethylbiguanide) belongs to guanidinium-derivative and is widely used for treatment of diabetic disorders in clinic. Metformin lowers blood glucose in diabetic animals through increase of glucose uptake into skeletal muscle. Recent evidence indicates that activation of imidazoline I2B receptor (I2BR) by guanidinium-derivatives also increased glucose uptake; however, the effect of metformin on I2BR is still unknown. The blood glucose levels were determined by a glucose kit. The ability of glucose uptake into isolated skeletal muscle or cultured C2C12 cells was determined using 2-[14C]-deoxyglucose as tracer. The expressions of 5' AMP-activated protein kinase (AMPK) and glucose transporter 4 (GLUT-4) were identified by Western blotting analysis. The metformin-induced blood glucose-lowering action was dose-dependently blocked by BU224, a specific I2R antagonist, in Wistar rats. Also, similar reversion by BU224 was observed in isolated skeletal muscle regarding the metformin-induced glucose uptake. Moreover, AMPK phosphorylation by metformin was concentration-dependently reduced by BU224 in isolated skeletal muscle. In addition, signals for metformin increased glucose uptake were identified via I2R/PI3K/PKC/AMPK dependent pathway in C2C12 cells. Thus, we suggest that metformin can activate I2BR to increase glucose uptake and I2BR will be a new target for diabetic therapy.     

AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells

This study examined the role of AMPK activation in osteoblast differentiation and the underlining mechanism. An AMPK activator (AICAR or metformin) stimulated osteoblast differentiation with increases in ALP and OC protein production as well as the induction of AMPK phosphorylation in MC3T3E1 cells. In addition, metformin induced the phosphorylation of Smad1/5/8 and expression of Dlx5 and Runx2, whereas compound C or dominant negative AMPK inhibited these effects. Transient transfection studies also showed that metformin increased the BRE-Luc and Runx2-Luc activities, which were inhibited by DN-AMPK or compound C. Down-regulation of Dlx5 expression by siRNA suppressed metformin-induced Runx2 expression. These results suggest that the activation of AMPK stimulates osteoblast differentiation via the regulation of Smad1/5/8-Dlx5-Runx2 signaling pathway.     

Nicotine-induced activation of AMP-activated protein kinase inhibits fatty acid synthase in 3T3L1 adipocytes: a role for oxidant stress

Recent studies suggest that the AMP-activated protein kinase (AMPK) acts as a major energy sensor and regulator in adipose tissues. The objective of this study was to investigate the role of AMPK in nicotine-induced lipogenesis and lipolysis in 3T3L1 adipocytes. Exposure of 3T3L1 adipocytes to smoking-related concentrations of nicotine increased lipolysis and inhibited fatty acid synthase (FAS) activity in a time- and dose-dependent manner. The effects of nicotine on FAS activity were accompanied by phosphorylation of both AMPK (Thr(172)) and acetyl-CoA carboxylase (ACC; Ser(79)). Nicotine-induced AMPK phosphorylation appeared to be mediated by reactive oxygen species based on the finding that nicotine significantly increased superoxide anions and 3-nitrotyrosine-positive proteins, exogenous peroxynitrite (ONOO(-)) mimicked the effects of nicotine on AMPK, and N-acetylcysteine (NAC) abolished nicotine-enhanced AMPK phosphorylation. Inhibition of AMPK using either pharmacologic (insulin, compound C) or genetic means (overexpression of dominant negative AMPK; AMPK-DN) abolished FAS inhibition induced by nicotine or ONOO(-). Conversely, activation of AMPK by pharmacologic (nicotine, ONOO(-), metformin, and AICAR) or genetic (overexpression of constitutively active AMPK) means inhibited FAS activity. Notably, AMPK activation increased threonine phosphorylation of FAS, and this effect was blocked by adenovirus encoding dominant negative AMPK. Finally, AMPK-dependent FAS phosphorylation was confirmed by (32)P incorporation into FAS in adipocytes. Taken together, our results strongly suggest that nicotine, via ONOO(-) activates AMPK, resulting in enhanced threonine phosphorylation and consequent inhibition of FAS.     

AMPK activation suppresses mTOR/S6K1 phosphorylation and induces leucine resistance in rats with sepsis

Although it has been known that protein synthesis is suppressed in sepsis, which cannot be corrected by leucine supplement (also known as leucine resistance), the molecular signaling mechanism remains unclear. This study aimed to investigate the AMP‐activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway in sepsis‐induced leucine resistance and its upstream signals, and to seek a way to correct leucine resistance in sepsis. Sepsis was produced by cecal ligation and puncture (CLP) model in rat. Both septic rats and sham operation rat received total parenteral nutrition (TPN) with or without leucine for 24 h, and then protein synthesis and AMPK/mTOR and protein kinase B (PKB) were tested. In vitro C2C12 cells were treated with or without leucine, and we tested the AMPK/mTOR pathway and protein synthesis. We blocked AMPK by compound C and stimulated it by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) individually. The results showed that AMPK was highly phosphorylated and suppressed mTOR/S6K1 activation in CLP rats. In vitro when AMPK was activated by AICAR, protein synthesis was suppressed and leucine resistance was observed. High phosphorylation of AMPK was accompanied by PKB inactivation in CLP rats. When PKB was blocked, both AMPK activation and leucine resistance were observed. In CLP rats, nutrition support with intensive insulin therapy reversed leucine resistance by activating PKB and suppressing AMPK phosphorylation. These findings suggest that high phosphorylation of AMPK induced by PKB inactivation in sepsis suppresses mTOR, S6K1 phosphorylation, and protein synthesis and leads to leucine resistance. Intensive insulin treatment can reverse leucine resistance by suppressing AMPK activation through activation of PKB.     

Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non–small cell lung cancer

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non–small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5′-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis–mediated survivin degradation, providing novel insights into the anticancer effects of metformin.     

Metformin protects against intestinal barrier dysfunction via AMPKα1‐dependent inhibition of JNK signalling activation

Disruption of the intestinal epithelial barrier, that involves the activation of C‐Jun N‐terminal kinase (JNK), contributes to initiate and accelerate inflammation in inflammatory bowel disease. Metformin has unexpected beneficial effects other than glucose‐lowering effects. Here, we provided evidence that metformin can protect against intestinal barrier dysfunction in colitis. We showed that metformin alleviated dextran sodium sulphate (DSS)‐induced decreases in transepithelial electrical resistance, FITC‐dextran hyperpermeability, loss of the tight junction (TJ) proteins occludin and ZO‐1 and bacterial translocation in Caco‐2 cell monolayers or in colitis mice models. Metformin also improved TJ proteins expression in ulcerative colitis patients with type 2 diabetes mellitus. We found that metformin ameliorated the induction of colitis and reduced the levels of pro‐inflammatory cytokines IL‐6, TNF‐a and IL‐1β. In addition, metformin suppressed DSS‐induced JNK activation, an effect dependent on AMP‐activated protein kinase α1 (AMPKα1) activation. Consistent with this finding, metformin could not maintain the barrier function of AMPKα1‐silenced cell monolayers after DSS administration. These findings highlight metformin protects against intestinal barrier dysfunction. The potential mechanism may involve in the inhibition of JNK activation via an AMPKα1‐dependent signalling pathway.     

Two Weeks of Metformin Treatment Enhances Mitochondrial Respiration in Skeletal Muscle of AMPK Kinase Dead but Not Wild Type Mice

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α₂ (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.     

Metformin induces Rab4 through AMPK and modulates GLUT4 translocation in skeletal muscle cells

Metformin is a major oral anti‐diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK‐dependent manner. Metformin stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the phosphorylation of PKC‐zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin‐induced phosphorylation of AS160/PKC‐zeta. In addition, a colorimetric absorbance assay showed that insulin‐induced translocation of GLUT4 was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC‐zeta but not with GLUT4. The C‐terminal‐deleted Rab4 mutant, Rab4ΔCT, showed diffuse sub‐cellular localization, while wild‐type Rab4 localized exclusively to the perinuclear membrane. Unlike Rab4ΔCT, wild‐type Rab4 co‐localized with PKC‐zeta. Together, these results demonstrate that metformin induces Rab4 expression via AMPK‐AS160‐PKC‐zeta and modulates insulin‐mediated GLUT4 translocation. J. Cell. Physiol. 226: 974–981, 2011. © 2010 Wiley‐Liss, Inc.